JPS59203564A - Self-antibody and immune composite adsorbing material - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides q! Regarding the adsorbent for body fluid evaporation that selectively adsorbs and removes autoantibodies and immune complexes that are thought to be closely related to type R diseases, it is particularly suitable for whole blood to which the direct overflow method is applied. Concerning suitable adsorbents.

As is well known, autoantibodies and/or immune complexes expressed in body fluids, such as blood, are associated with cancer, proliferative syndromes, and autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, as well as cancer, VJ legis, and organs. It is believed that there is a close relationship with the cause or progression of diseases and phenomena related to the body's immune function, such as rejection reactions during transplantation.

Therefore, from body fluid components such as blood and plasma, the above autoantibodies and/or immune-like combinations are detected. It was expected that by differentially adsorbing and removing it, it would be possible to prevent the progression of the above-mentioned diseases, alleviate symptoms, and even hasten healing.

The present inventors have discovered that autoantibodies and/or immune complexes can be selectively adsorbed with high efficiency, non-selective adsorption is low, safety is achieved, and sterilization can be easily carried out.
As a result of our research with the aim of providing an adsorbent suitable for purification or regeneration of body fluids, we found that the carrier retains a substance that destroys the special chemical structure that forms a selective chemical interaction with the adsorbed substance. He discovered various adsorbents and filed patent applications (% patent application No. 7152/1980, % patent application No. 18923/1982, Japanese patent application No. 56/76776, Japanese patent application No. 56/15944).

The non-invention is related to the previous invention, and is a result of a detailed study of the substance that selectively interacts with the adsorbed substance, and relates to an improvement of the above-mentioned adsorbent.

Desired properties of the adsorbent used for the purpose of the present invention are (11. VC that is selective for all autoantibodies and immune complexes, and that it adsorbs with a transparent moving part; (21. (31) ability to easily activate the coagulation and fibrinolytic system, (4) ability to sterilize, (5) mechanical strength and ability to separate light.

The present defenders have proposed that, as an eM material for body fluid purification, it maintains adsorption performance capable of adsorbing all autoantibodies and immune complexes with high efficiency, and also has low C1 non-selective adsorption, i.e. autoantibodies and/or immune complexes. We are conducting further intensive research with the goal of providing an adsorbent that can adsorb external biological components with less force and less activation of the coagulation fibrinolytic system.
I fell asleep.

Various compounds were immobilized on various carriers and the adsorption properties for autoantibodies and/or immune complexes and non-selective adsorption properties for coexisting proteins were evaluated. An adsorbent in which a low-molecular-weight compound with a skeleton structure of 6 or more carbon atoms is immobilized on a single coral body adsorbs autoantibodies and immune complexes17, t
They also found that proteins useful to living organisms, such as fifurinogen, become difficult to adsorb, and furthermore, they found that when the molecular weight of a compound immobilized on a carrier is relatively small, even if the compound is detached from the carrier and becomes difficult to adsorb to living organisms. The present invention was made based on the 4'' suspicion that it does not have antigenicity even if it enters the cellulose.

That is, the present invention provides an autoantibody and/or immunocomplex in which an organic low-molecular compound having at least 6 carbons in its backbone structure is bound to an insoluble organic phase having a negative charge. As for the adsorbent, the protein exclusion limit molecular weight of the carrier is preferably in the range of 150,000 to 10,000,000.

In the present invention, the negatively charged insoluble organic carriers include carboxylmethyl-polyacrylamide-based carriers, aminohexylsuccinylated cellulose, sulfoethyl-cellulose, phosphate ester-cellulose, carboxyhexyl-agarose, etc., and styrene divinylbenzene. The carrier itself exhibits a single negative charge in neutral electrolyte fluids such as blood and body fluids, such as ion exchange resins in which cation exchange groups such as sulfone groups and carboxyl groups are bonded to a polymeric base. say. Alternatively, a carrier that does not have a negative charge may be coated with a film that has a negative charge. An organic low-molecular compound having at least 6 carbons in its skeleton structure is
The number of carbon atoms in the skeleton structure is 6 or more, and the molecular weight is 1.
04 or less, preferably 103 or less. Among these, ffl-like compounds with aromatic properties give good results.

Any compound with aromatic properties can be usefully used, but benzene-based aromatic rings such as benzene, naphthalene, and phenanthrene, oxygen-containing aromatic rings such as dibenzofuran, fromene, and benzofuran, and thianaphthene, titentrene, etc. Sulfur-containing aromatic ring, phenanthrene, kino 1
Aromatic ring forces such as nitrogen-containing 6-membered rings such as non, aku1-nodine, etc., and nitrogen-containing 5-membered rings such as indole, carnoxole, etc.;
Gives good results. Among these, benzene aromatic rings give good results in %.

The number of carbon atoms in the skeleton structure of an organic low-molecular compound as used in the present invention refers to all carbons contained in the organic low-molecular compound, excluding carbons of carboxyl groups. Here, all the carbon atoms in the carboxyl group were removed because the carboxyl group is hydrophilic and exhibits only a negative and small charge effect. The number of carbon atoms in substituents other than carboxyl, i.e.
Carbons contained in alkoxyl groups, aldehyde groups, alkoxycarbonyl groups, etc. are counted.

Carbon contained in organic low-molecular compounds and self-destruction, body and/or
Alternatively, hydrophobic interaction force (van der Waals force) acts between the immune complex and the immune complex.

Negatively charged insoluble organic carrier e) Coulomb force and hydrophobic interaction force of organic low-molecular compounds (by working synergistically, autoantibodies and/or While maintaining this, it becomes possible to suppress non-selective adsorption of components useful to living organisms such as fibrinogen.

The larger the number of negatively charged functional groups on the negatively charged insoluble organic carrier, the higher the ability to adsorb autoantibodies and/or immune complexes, and the non-selective absorption of proteins useful to living organisms such as fibrinogen. Adsorption is suppressed. The functional group site showing a negative charge has a unit surface area of 0.0
It is preferably 2 μmol/m2 or more, more preferably 0.2 μm o 17 m” to 200 pmol/m
rn2, more preferably 2 μmol/m2 to 200
μrnol 7m2.

The greater the number of carbon atoms in the skeleton structure of the organic low-molecular-weight compound that binds to the insoluble organic carrier that carries the negative charge, the more likely it is that autoantibodies and/or
Although the adsorption capacity of the immune complex increases, the non-selective adsorption of proteins such as fibrinogen also increases. This is because Coulomb force plays an essential role in improving adsorption selectivity in the interaction between proteins and organic low-molecular compounds bound to negatively charged insoluble organic carriers9. This means that negative charges play an important role in improving the adsorption selectivity of fibrinogen, a coexisting protein, while maintaining the adsorption ability of autoantibodies and/or immune complexes. .

In the present invention, the number of carbon atoms in the skeleton structure of the organic low molecular compound bonded to the negatively charged insoluble organic carrier is 6 or more, which means that the adsorption ability of autoantibodies and/or immune complexes is improved. In the case of carbon having an unsaturated bond, the number is preferably 6 or more, preferably 10 or more, and in the case of carbon having only a single bond, the number is preferably 10 or more, and preferably 15 or more. Further, the upper limit is determined by the molecular weight, but the number of carbon atoms is preferably 500 or less, and preferably 50 or less. When the +I compound is a specific compound having a hydrophobic substituent such as chlorine or iodine, the adsorption characteristics of the target adsorbent substance are improved compared to a compound having no substituent.

On the other hand, organic compounds with many non-dissociable (hydrophilic) substituents such as hydroxyl groups, thiol groups, and amide groups are preferable because their adsorption to the target adsorbent is lower than those without substituents. do not have. The number of non-dissociable hydrophilic substituents is preferably less than 1 per 2 carbon atoms in the skeletal structure of the organic compound, and desirably less than 1 per 5 carbon atoms. This is thought to be due to the difference in the mutual hydrophobic rh utility exerted on the target adsorbent substance depending on the bonding mode of carbon, type of substituent, etc.

The molecular weight of the organic compound that binds to the negatively charged insoluble organic carrier is preferably 104 or less from the viewpoint of antigenicity even if the bond is broken and enters the living body. More preferably it is 103 or less.

As the negatively charged insoluble organic carrier used in the present invention, either a hydrophilic carrier or a hydrophobic carrier can be used, but when a hydrophobic carrier is used, nonspecific adsorption of albumin to the carrier sometimes occurs. The hydrophilic 11-body gives more favorable results.

The shape of the insoluble organic substance carrier having a negative charge may be any known shape such as particulate, fibrous, hollow fiber, or membrane. From the viewpoint of ease of handling as an adsorbent, particles or fibers are preferable.

The average particle size of the spherical or particulate carrier is 25 to 2500 μm
However, its specific surface area (adsorption capacity as an adsorbent) and flow rate of body fluids are 3r! From i-side, 50-1500
Particularly preferred is one in μm.

The specific surface area of the carrier is preferably 5 m2/f or more, and 55 i
n2/7 or more is desirable.

Porous particles, especially porous polymers, can also be used as particulate carriers. The porous polymer particles used in the present invention have a large number of negatively charged functional groups in the surface m1 and are capable of immobilizing an organic low molecular compound having at least 6 carbons in the skeleton structure. The exclusion limit molecular weight (protein) is 150,000 to 10,000,000 for immune complexes, especially IgM immune complexes, since the molecular weight of the target adsorbent of the present invention is 150,000 (IgG).
0 million is preferred. The most common exclusion limit molecular weight for purposes of this invention is 1 million to 5 million.

When a fibrous carrier is used, the diameter of the fibers is preferably in the range of 0.02 tenier to 10 deniers, more preferably 0.1 tenier to 5 deniers. If the fiber diameter is too large, the adsorption amount and rate of globulin-based compounds will be reduced, and if it is too small, activation of coagulation threads, blood cell adhesion, and clogging are likely to occur.

Methods for bonding an organic low-molecular compound having at least 6 carbons in its skeleton structure to an insoluble organic substance having a negative charge include covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation on the surface of subaggregates. Any known method can be used, but in view of the elution properties of the bound substance, it is most preferable to use it after it is immobilized and insolubilized by covalent bonding. Therefore, it is possible to use the binding method of the ligand normally used in immobilized stem material and Affinikei chromatography.

Further, an organic compound may be covalently bonded using a part of the negatively charged functional group.

The material 1 & of the present invention can be used by being filled and held in a container equipped with an inlet/outlet for body fluids.

In the drawings, reference numeral 1 shows an example of the l&adsorption device using the adsorbent for autoantibodies and/or immune complexes according to the present invention, in which a backing with a filter 6 lined inside the cylinder 2 is attached to an open BIS at one end. A cap 6 having a body fluid inlet 5 is fitted with a screw through the cap 6, and a body fluid outlet lower is inserted into the opening at the other end of the cylinder 2. A cap 8 is screw-fitted to form the entire container, and an adsorbent layer 9 is formed by filling and holding an adsorbent into the gap between the filters 32 and 6'.

The adsorbent layer 9 may be filled with the adsorbent of the present invention alone, mixed with other adsorbents, or in a Fift layer.

Other adsorption amounts include other malignant substances such as DNA (
It is possible to use activated carbon, which has a wide range of adsorption capacities, and adsorption of antigens). As a result, a wider range of clinical applications can be expected due to the synergistic effect of the adsorbent. When used for extracorporeal circulation, the volume of the adsorbent layer 9 is increased by about 50 to 400 roux.

There are two methods for using the device of the present invention in extracorporeal circulation, as shown below. One is to use a centrifuge or a model plasma separator to extract blood from the body.
After separating plasma components and blood cell components, the plasma components are passed through the device, purified, and then returned to the body together with the blood cell components. This is a method of purifying the equipment by passing it through a groove.

Regarding the passage speed of blood or plasma through the casing, the adsorption efficiency of the adsorbent is very high, so the particle size of the adsorbent can be made coarser, and the degree of packing can be lowered, so it is possible to change the shape of the adsorbent layer. A high passing speed can be provided regardless of the situation. Therefore, a large amount of body ridge treatment can be performed.

The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or equipment conditions.

As described above, the adsorbent of the present invention selectively adsorbs and removes autoantibodies and/or immune complexes in body fluids at a high rate, allows the construction of a very compact adsorption device, and is non-selective. Since it has little adsorption, it is easy and safe to use.

9B1 of the present invention. The lid material can be used for general purification and regeneration of body fluids such as autologous plasma, autologous blood, cancer, etc.
Immunoproliferative syndrome, rheumatoid arthritis, collagen diseases such as systemic lupus erythematosus, autoimmune diseases such as myasthenia gravis,
It can be effectively used for extracorporeal circulation treatment of diseases and phenomena related to the body's immune function such as allergies and rejection reactions during organ transplants, kidney diseases such as nephritis, and liver diseases such as hepatitis.

Furthermore, since an insoluble organic substance enclosure having a negative charge is used, adhesion and aggregation of platelets are suppressed.

Therefore, it can be effectively used as an extracorporeal circulation treatment device for whole blood.

Embodiments of the present invention will be described in detail below using Examples. .

Example 1 Carboxyhexyl-Sepharose 4B (manufactured by Pharmacia, Sweden) was used as the insoluble 11-carrier having a negative charge, and an organic compound having 6 or more carbons in the skeleton structure was prepared by the usual method of reacting carbodiimide with this carrier. At this time, the activation rate by carbodiimide was kept low so that about half of the negatively charged functional groups remained. Excess active groups were blocked with ethanolamine. The amount of organic compounds retained is determined by converting the primary amino group of the remaining organic compound into 4-phenylspiro[furan-2(3H
), 1'-phthalane:] -] 3.3'-dione "Flurum" (manufactured by Roche), and the identification rate was adjusted to 2 ln9/ml (press body).

The adsorption experiment was carried out using A, 3 volumes of Umachi Soybean Colony added to CD, and G&
After thoroughly mixing with 10 volumes of light material and incubating at 57C for 3 hours, the supernatant plasma was evaluated.

Evaluations were made for rheumatoid factor, 1 immune complex, fibrinogen, and immunoglobulin G.

Rheumatoid factor was measured using a latex agglutination test and a passive sensitization hemagglutination test.

The latex enzyme test is a detection method that measures the property of latex particles agglutinating when human γ-globulin is adsorbed onto polystyrene latex particles and Eisha plasma containing rheumatoid factor is applied. A plasma dilution series is created and the rheumatoid factor concentration is evaluated at the plasma dilution rate at which latex particles no longer aggregate.

Plasma containing a high concentration of rheumatoid factor will have a high dilution rate to become negative, whereas plasma with a low concentration will have a low dilution.

A passive sensitization hemagglutination test was performed using rabbit γ-γ to sheep erythrocytes.
- Globulin is adsorbed, and the rest is the same as the latex agglutination test. In general, it is said that the passivity f/F = collapsing ball agglutination test has a higher rheumatoid factor ratio than the latex agglutination test.

A dilution series was prepared with glycine saline buffer, and the dilution height at which rheumatoid factor became negative in the latex agglutination test was determined. The latex@collection test was conducted using a kit from Japan Freeze Drying Research Institute. Similarly, passive sensitization hemagglutination test)
(RAHA test, manufactured by Fuji Kinki Soyaku ■) was evaluated.

Immune complexes were prepared by polyethylene glycol precipitation. In this method, the amount of immune complexes is determined by quantifying the amount of immunoglobulin using a single radial immunodiffusion method of the immune complexes precipitated and fractionated with polyethylene glycol. The operating method and φ items of this method are as follows.

(11 samples 1. Place in Odi test tube, 8% PEG (polyethylene glycol, average molecular weight 6000-7500)
Add 1.0 g of, stir, and leave at 4C for 60 minutes.

+2+4 tl', 1ooo, centrifuge for 60 minutes, and remove the supernatant. After removal, the obtained precipitate was P B S (!J
Redissolve in acid buffered saline to make a total volume of 1°01 nl.

(31111, (2+) is repeated two more times to wash away any contaminating monomeric immunoglobulin.

(4) @PBS suspension of the finally obtained immune complex was subjected to single radial immunodiffusion method.
Quantify immunoglobulin G.

The amounts of fibrinogen and immunoglobulin G were determined by the single radial immunodeficiency method.

The values of the rheumatoid patient's plasma used are: rheumatoid factor latex 160, RAHA 640, immune complex (measures immunoglobulin G) 45 m:2/di, fibrinogen 28011197 dt% immunoglobulin G 1280 m9/d7 Met.

The results of the adsorption experiment are shown in Table 1. From Table 1, an adsorbent in which an organic low-molecular compound having at least 6 carbons in its skeleton structure is bound to an insoluble organic press having a negative charge selectively adsorbs rheumatoid factors, immune complexes + bs, and fibrinogen. , it can be seen that there is little non-selective adsorption of immunoglobulin G.

Table 1 Comparative Example 1 * 'iQ, Sepharose 4B as a non-responsive partner
(manufactured by Pharmacia, Sweden) was activated with CNBr in a conventional manner, and then β-phenyl-ethylami/ was immobilized. The number of carbon atoms of β-phenyletheramine was 8, and the amount of immobilization was 2.1 to ml (q', body of the eye).

The adsorption experiment was conducted in the same manner as in Example 1 using the same plasma as in Example 1.

As a result, the RA FI A titer went from 640 to 320.
The latex titer has dropped from 160 to 80, the immune complex has dropped from 45 to 28 ■/dt, but the fibrinogen has dropped to 2.
80 has dropped to 175 rn9/d7.

[Brief explanation of the drawing]

The drawing is a schematic diagram showing an example of an adsorption device in which a container is filled with the adsorbent of the present invention.

Claims (1)

[Claims] (1) An adsorbent for autoantibodies and/or immune complexes, characterized in that an organic low-molecular compound having at least 6 carbon atoms in its skeleton structure is bonded to a negatively charged insoluble organic carrier. (The adsorbent for autoantibodies and/or immune complexes according to claim 1, wherein the insoluble organic carrier having a 21 negative charge has a protein exclusion limit molecular weight in the range of 150,000 to 10,000,000.